The overall objective of these studies is to investigate the regulation of smooth muscle contraction by the thin filament-linked regulatory protein caldesmon and to determine how the interplay between thin-filament-linked and thick-filament-linked systems regulates the level of cross bridge activation (force production) and the rate of cross bridge cycling (unloaded shortening velocity). The proposed studies will test the general hypothesis that the actin-binding protein, caldesmon, is a major component of an actin-linked regulatory system which modifies the activation of contraction which occurs in response to myosin light chain phosphorylation. The proposed studies will specifically test the hypotheses that 1) caldesmon modulates both cross bridge cycling rates and the number of attached cross bridges at any given level of myosin phosphorylation, 2) that both calcium-calmodulin and caldesmon phosphorylation regulate the effects of caldesmon, and 3) that caldesmon is localized to a subpopulation of the actin filaments in smooth muscle. Using rat uterine smooth muscle as an experimental model, we will (Specific Aim 1) demonstrate reversible phosphorylation of caldesmon in intact tissues, (Specific Aim 2) determine if caldesmon kinase is protein kinase C or CaM kinase II, (Specific Aim 3 and 4) determine a) if caldesmon regulates force production and/or shortening velocity during contraction, b) if caldesmon-dependent regulation is controlled by Ca/CaM and/or phosphorylation, and c) if caldesmon acts to caldesmon on native thin filaments isolated from fast, phasic and slow, tonic smooth muscles, and (Specific Aim 6) demonstrate that models of caldesmons regulation derived from permeabilized muscle studies are consistant with the changes in caldesmons phosphorylation, force development and shortening velocity measured in pharmacologically activated smooth muscles. The experimental approach will involve the measurement of caldesmon phosphorylation, force development, and isotonic shortening velocity during contractions of both intact and chemically permeabilized uterine smooth muscle. The levels of caldesmon and myosin light chain phosphorylation will be manipulated in chemically permeabilized (skinned) muscles while measuring the effects on contractile properties (i.e. shortening velocity and force development); caldesmon phosphorylation will be altered using purified kinases, phosphatase, and synthetic peptide inhibitors of protein kinase C and CaM kinase II. Phosphopeptide mapping, phosphoamino analysis, and gas phase microsequencing will be used to analyze the phosphorylation sites on caldesmon and to identify caldesmon kinase. Lastly, the distribution of caldesmon among native actin filaments isolated from tonic, vascular, and phase nonvascular smooth muscle by immunoprecipitation with anticaldesmon antibodies will determine if there are caldesmon-free populations of actin in smooth muscles. These studies will not only verify that caldesmon plays a regulatory role in smooth muscle but will also provide significant new information about how caldesmon participates in the regulation of the latch-state in smooth muscle.